Protein with recombinase activity for site-specific DNA-recombination

ABSTRACT

The invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination and a method for producing a site-specific DNA recombination. The invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medical research. The objective of the invention is solved by the use of a protein with recombinase activity to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other. The invention also includes a method for producing a site-specific DNA recombination comprising the steps of a) providing a cell comprising at least two recognition sites that are identical or reverse complementary to each other; and b) contacting a protein with recombinase activity with the recognition sites, thereby producing the site-specific DNA-recombination.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:28460020003_sequence_listing.txt; Size: 63.3 kilobytes; and Date ofCreation: Jun. 26, 2017) filed with the application is incorporatedherein by reference in its entirety.

The invention relates to the use of a protein with recombinase activityto catalyze a site-specific DNA recombination and a method for producinga site-specific DNA recombination. The invention is applicable alone orin combination with other recombinase systems for genetic manipulation,for example in medical research.

The use of site-specific DNA recombinases allows genetic manipulationsin both prokaryotic and eukaryotic organisms. For this purpose, varioussite-specific DNA recombinases isolated from different organisms areused. The DNA recombination mediated by the site-specific recombinaseoccurs by cleavage and rejoining of DNA at specific DNA sequences, theso-called recognition sites (nucleic acid sequences of 10 to 150 basepairs). If two recognition sites are oriented in the same direction in aDNA strand, a nucleic acid segment flanked by the recognition sites iscut out (excision). If two recognition sequences flanking a nucleic acidsegment in a DNA strand are oriented in the opposite direction, thesite-specific DNA recombinase catalyzes the inversion of the nucleicacid segment. If two recombination sites are located on two differentmolecules, then the site-specific DNA recombinase catalyzes merge of twomolecules (integration).

Among sites-specific recombinases a particular class called tyrosinerecombinases (SSRs), such as Cre and Flp, has become an outstandinggenetic tool. Unlike most SSRs, they do not require additional hostfactors for efficient catalysis and recognize relatively shortsequences. Because of the simplicity and efficiency, these recombinasesnow serve as “molecular scissors” for robust, non-disruptive andreproducible genomic modifications.

The so-called Cre/loxP system (EP 0 2200 009 B1) is widely used in theprior art. Cre (amino acid sequence according to SEQ ID No. 4) is asite-specific DNA recombinase isolated from bacteriophage P1. Therecognition site of the Cre protein is a nucleotide sequence of 34 basepairs, the so-called loxP site (SEQ ID No. 5). Cre shows highrecombinase activity both in bacterial and mammalian cells. It is knownto modify the amino acid sequence of Cre in order to obtain novelsite-specific recombinases (DE 102 07 313 A1).

Another site-specific DNA recombinase system is the so-called Flp/FRTsystem isolated from Saccharomyces cerevisiae. The Flp/FRT systemincludes the recombinase Flp (flippase) (amino acid sequence accordingto SEQ ID No. 6) that catalyzes DNA-recombination on its recognitionsites, the so-called FRT sites (SEQ ID No. 7).

In addition to the Cre/loxP and the Flp/FRT system, that are both themost widely used site-specific recombinase systems of tyrosine class,other recombinase systems are known in the art. U.S. Pat. No. 7,422,889and U.S. Pat. No. 7,915,037 B2 disclose the so-called Dre/rox systemthat comprises a Dre recombinase (amino acid sequence according to SEQID No. 8) isolated from Enterobacteria phage D6, the recognition site ofwhich is called rox-site (SEQ ID No. 9). Further known recombinasesystems are the VCre/VloxP system isolated from Vibrio plasmid p0908(amino acid sequence according to SEQ ID No. 10 for the recombinase, andSEQ ID No. 11 for the VloxP site), and the sCre/SloxP system (WO2010/143606 A1; Suzuki and Nakayama, 2011).

The known recombinase systems that are known in the art show differentactivities in cells of different origin. For many applications, such asthe production of transgenic animals with conditional gene knockouts,two or more recombinase systems are used in combination with each other.However, emerging complex genetics studies and applications requiresimultaneous use of multiple recombinases. At the same time not allwell-described site-specific recombinases are equally applicable in allmodel organisms due to, e.g. genome specificity (off-target activity oncryptic recognition target sites). For that reason it is important thatan optimal recombinase can be chosen depending on the target organism orexperimental setup. Therefore, there is a need for the provision ofalternative recombinase systems that can be used to catalyze asite-specific DNA recombination on short targets in a variety of celltypes and with high activity and with low toxicity. It is the objectiveof the invention to provide novel recombinase systems for site-specificgenetic recombination that can be used in a variety of cell types.Another object of the invention is to provide a novel, highly specific,recombinase system for site-specific genetic recombination withpreferably low toxicity.

The objective is solved by the use of a protein with recombinaseactivity, wherein the protein comprises an amino acid sequenceexhibiting at least 70%, preferably at least 80%, preferably at least90%, particularly preferred at least 95%, even more preferred at least99% amino acid sequence identity to SEQ ID No. 1 to catalyze asite-specific DNA recombination at, preferably at two, recognition sitesthat are identical or reverse complementary to each other, wherein atleast one recognition site comprises a nucleic acid sequence accordingto or reverse complementary to SEQ ID No. 2; or a nucleic acid sequencethat is a functional mutant thereof.

The protein with recombinase activity as defined above and used in theinvention protein is referred to herein as “Vika”. The recognition siteof the site-specific recombinase Vika is referred to herein as“vox-site” or simply “vox”. A vox-site is characterized by its nucleicacid sequence according to SEQ ID No. 2 or a nucleic acid sequencereverse complementary thereto. Recognition sites that exhibit a nucleicacid sequence identity to SEQ ID No. 2 of at least 70%, preferably atleast 80%, preferably at least 90%, particularly preferred at least 95%,even more preferred at least 99%, or nucleic acid sequences reversecomplementary thereto; and that are targets for the site specificrecombination of Vika (herein referred to as “functional mutants” of voxsites) are also “vox-sites” within the sense of the invention. However,particularly preferred vox-sites exhibit a nucleic acid sequenceaccording to SEQ ID No. 2 or a nucleic acid sequence reversecomplementary thereto (herein also referred to as “wild type vox-site”).

By a functional mutant of the wild type vox-site is meant that one ormore nucleic acids are added to, inserted, deleted or substituted fromthe nucleic acid sequence according to SEQ ID No. 2. At the same time,the functional mutant of the recognition site with a nucleic acidsequence according to SEQ ID No. 2 exhibits a nucleic acid sequenceidentity to SEQ ID No. 2 of at least 70%, preferably at least 80%,preferably at least 90%, particularly preferred at least 95%, even morepreferred at least 99% and is a functional recognition site of Vika.Preferred mutations are point mutations or an exchange of the spacerregion of the recognition site according to SEQ ID No. 2. It is knownthat the exchange of a spacer region of a recognition site does notinfluence its activity as a target site for the specific recombinase.Therefore, particularly preferred functional mutants of the wild typevox-site exhibit a nucleic acid sequence identity of at least 70%,preferably at least 75%, to the nucleic acid sequence according to SEQID No. 2 or its reverse complementary sequence; and exhibit mutations inthe nucleic acids 14 to 21 (spacer region) of SEQ ID No. 2.

The invention is based on the finding of the inventors that the proteinZP_05884863 (www.ncbi.nlm.nih.gov/protein/ZP_05884863, SEQ ID No. 1),which is referred to herein as Vika (wild type), shows a Crerecombinase-like activity and recognizes recognition sites according toSEQ ID No. 2. Vika is derived from the gram-negative bacterium Vibriocoralliilyticus ATCC BAA 450. The amino acid sequences of Cre and Vikashow a low identity of merely 26% (49% sequence similarity). Therecognition sequences for Cre and Vika show a low identity of 35.6%(35.6% sequence similarity).

The inventors identified six putative recognition sites with a lox-likestructure in Vibrio coralliilyticus. Upon extensive studies only onethereof, the wild type vox site, turned out to be the actual recognitionsite of Vika. The wild type vox-site is a 34 bp region of two invertedrepeats that comprises about 50% sequence homology to the loxP site. Byexpression of the nucleic acid sequence encoding for Vika (SEQ ID No. 3)in E. coli, the Vika protein could be successfully obtained and itsrecombinase activity and specifity for the wild type vox site could bedemonstrated. Vika was shown to belong to tyrosine class of SSRs andtherefore, as further demonstrated in experiments, does not requireexpression of auxiliary factors for enzymatic activity in various cellstypes. Furthermore, it was shown that Vika does not cross react withother known recombinase systems and is superior in the activity comparedto some of the known recombinase systems, at least in certain celltypes.

The invention is based further on the finding that the proteinWP_008927019.1 (www.ncbi.nlm.nih.gov/protein/WP_008927019.1, SEQ ID No.35), which is referred to herein as Panto (wild type), shows a Crerecombinase-like activity and recognizes recognition sites according toSEQ ID No. 36. Panto is derived from the enterobacterium Panteo sp. aB.The amino acid sequences of Cre and Panto show a low identity of 41%(57% sequence similarity).

The invention is based further on the finding that the proteinYP_004250912. (www.ncbi.nlm.nih.gov/protein/YP_004250912.1, SEQ ID No.37), which is referred to herein as Nigri (wild type), which was alreadypredicted to be a putative Cre-like recombinase, recognizes recognitionsites according to SEQ ID No. 38. Nigri is derived from thegram-negative bacterium Vibrio nigripulchritudo. The recognitionsequences for Cre and Nigri show a low identity of 34.7% (34.7% sequencesimilarity).

The inventors identified recognition sites for both Panto and Nigri.

The objective is further solved by the use of a protein with recombinaseactivity, wherein the protein comprises an amino acid sequenceexhibiting at least 70%, preferably at least 80%, preferably at least90%, particularly preferred at least 95%, even more preferred at least99% amino acid sequence identity to SEQ ID No. 35 to catalyze asite-specific DNA recombination at, preferably at two, recognition sitesthat are identical or reverse complementary to each other, wherein atleast one recognition site comprises a nucleic acid sequence accordingto or reverse complementary to SEQ ID No. 35; or a nucleic acid sequencethat is a functional mutant thereof.

The protein with recombinase activity as defined above and used in theinvention protein is referred to herein as “Panto”. The recognition siteof the site-specific recombinase Panto is referred to herein as“pox-site” or simply “pox”. A pox-site is characterized by its nucleicacid sequence according to SEQ ID No. 36 or a nucleic acid sequencereverse complementary thereto. Recognition sites that exhibit a nucleicacid sequence identity to SEQ ID No. 36 of at least 70%, preferably atleast 80%, preferably at least 90%, particularly preferred at least 95%,even more preferred at least 99%, or nucleic acid sequences reversecomplementary thereto; and that are targets for the site specificrecombination of Panto (herein referred to as “functional mutants” ofpox sites) are also “pox-sites” within the sense of the invention.However, particularly preferred pox-sites exhibit a nucleic acidsequence according to SEQ ID No. 36 or a nucleic acid sequence reversecomplementary thereto (herein also referred to as “wild type pox-site”).

By a functional mutant of the wild type pox-site is meant that one ormore nucleic acids are added to, inserted, deleted or substituted fromthe nucleic acid sequence according to SEQ ID No. 36. At the same time,the functional mutant of the recognition site with a nucleic acidsequence according to SEQ ID No. 36 exhibits a nucleic acid sequenceidentity to SEQ ID No. 36 of at least 70%, preferably at least 80%,preferably at least 90%, particularly preferred at least 95%, even morepreferred at least 99% and is a functional recognition site of Panto.Preferred mutations are point mutations or an exchange of the spacerregion of the recognition site according to SEQ ID No. 36. It is knownthat the exchange of a spacer region of a recognition site does notinfluence its activity as a target site for the specific recombinase.Therefore, particularly preferred functional mutants of the wild typepox-site exhibit a nucleic acid sequence identity of at least 70%,preferably at least 75%, to the nucleic acid sequence according to SEQID No. 36 or its reverse complementary sequence.

The objective is further solved by the use of a protein with recombinaseactivity, wherein the protein comprises an amino acid sequenceexhibiting at least 70%, preferably at least 80%, preferably at least90%, particularly preferred at least 95%, even more preferred at least99% amino acid sequence identity to SEQ ID No. 37 to catalyze asite-specific DNA recombination at, preferably at two, recognition sitesthat are identical or reverse complementary to each other, wherein atleast one recognition site comprises a nucleic acid sequence accordingto or reverse complementary to SEQ ID No. 37; or a nucleic acid sequencethat is a functional mutant thereof.

The protein with recombinase activity as defined above and used in theinvention protein is referred to herein as “Nigri”. The recognition siteof the site-specific recombinase Nigri is referred to herein as“nox-site” or simply “nox”. A nox-site is characterized by its nucleicacid sequence according to SEQ ID No. 38 or a nucleic acid sequencereverse complementary thereto. Recognition sites that exhibit a nucleicacid sequence identity to SEQ ID No. 38 of at least 70%, preferably atleast 80%, preferably at least 90%, particularly preferred at least 95%,even more preferred at least 99%, or nucleic acid sequences reversecomplementary thereto; and that are targets for the site specificrecombination of Nigri (herein referred to as “functional mutants” ofnox sites) are also “nox-sites” within the sense of the invention.However, particularly preferred nox-sites exhibit a nucleic acidsequence according to SEQ ID No. 38 or a nucleic acid sequence reversecomplementary thereto (herein also referred to as “wild type nox-site”).

By a functional mutant of the wild type nox-site is meant that one ormore nucleic acids are added to, inserted, deleted or substituted fromthe nucleic acid sequence according to SEQ ID No. 38. At the same time,the functional mutant of the recognition site with a nucleic acidsequence according to SEQ ID No. 38 exhibits a nucleic acid sequenceidentity to SEQ ID No. 38 of at least 70%, preferably at least 80%,preferably at least 90%, particularly preferred at least 95%, even morepreferred at least 99% and is a functional recognition site of Nigri.Preferred mutations are point mutations or an exchange of the spacerregion of the recognition site according to SEQ ID No. 38. It is knownthat the exchange of a spacer region of a recognition site does notinfluence its activity as a target site for the specific recombinase.Therefore, particularly preferred functional mutants of the wild typenox-site exhibit a nucleic acid sequence identity of at least 70%,preferably at least 75%, to the nucleic acid sequence according to SEQID No. 38 or its reverse complementary sequence.

Within the sense of the invention the terms “site-specific DNArecombinase” and “recognition site” are used as defined above in thediscussion of the prior art.

The invention also includes a method for producing a site-specific DNArecombination. The method according to the invention comprises the stepsof

-   a) providing a cell comprising at least two recognition sites that    are identical or reverse complementary to each other, wherein at    least one recognition site comprises a nucleic acid sequence    according to or reverse complementary to SEQ ID No. 2; or a nucleic    acid sequence that is a functional mutant thereof; and-   b) contacting a protein with recombinase activity, wherein the    protein exhibits an amino acid sequence of at least 70%, preferably    at least 80%, preferably at least 90%, particularly preferred at    least 95%, even more preferred at least 99%, amino acid sequence    identity to SEQ ID No. 1 with the recognition sites, thereby    producing the site-specific DNA-recombination.

In this method according to the invention the Vika protein is contactedwith at least two vox sites preferably inside a cell. Upon binding ofthe Vika protein to the vox sites, site-specific DNA-recombinationoccurs.

The invention further includes a method for producing a site-specificDNA recombination using Panto or Nigri. The method according to theinvention comprises the steps of

-   a) providing a cell comprising at least two recognition sites that    are identical or reverse complementary to each other, wherein at    least one recognition site comprises a nucleic acid sequence    according to or reverse complementary to SEQ ID No. 36 or 38; or a    nucleic acid sequence that is a functional mutant thereof; and-   b) contacting a protein with recombinase activity, wherein the    protein exhibits an amino acid sequence of at least 70%, preferably    at least 80%, preferably at least 90%, particularly preferred at    least 95%, even more preferred at least 99%, amino acid sequence    identity to SEQ ID No. 35 or 37 with the recognition sites, thereby    producing the site-specific DNA-recombination.

In this method according to the invention the Panto protein is contactedwith at least two pox sites inside a cell. Alternatively the Nigriprotein is contacted with at least two nox sites inside a cell. Uponbinding of the Panto or Nigri protein to the pox sites or respectivelyto the nox sites, site-specific DNA-recombination occurs.

The method according to the invention can be carried out in vitro or invivo. In case the invention is carried out in an animal (includinghumans) it is preferably carried out for non-therapeutic use. The methodis applicable in all areas where state of the art site specificrecombinases are conventionally used (including inducible knock out orknock in mice and other transgenic animal models). In preferred methodsaccording to the invention, the site-specific recombination results inintegration, deletion, inversion, translocation or exchange of DNA.Preferably, the method according to the invention is not used for thetherapeutic treatment of a human being or an animal. Preferably themethod according to the invention is used to create animal models, whichare useful for biomedical research, e. g. as models for human diseases.

In a method according to the invention, the nucleic acid sequenceencoding for Vika (or Panto or Nigri respectively) is either alreadypresent in the cell or introduced into the cell, preferably byrecombinant techniques. This preferred method according to the inventionfurther includes the step of

-   c) introducing into the cell a nucleic acid encoding for the Vika    protein (or the Panto or Nigri protein respectively) with    recombinase activity, wherein said nucleic acid encoding for the    Vika protein preferably exhibits at least 70%, preferably at least    80%, preferably at least 90%, particularly preferred at least 95%,    even more preferred at least 99% nucleic acid sequence identity to    SEQ ID No. 3.

For activation of the expression of the nucleic acid encoding for Vika(or Panto or Nigri respectively), the nucleic acid encoding for Vika (orPanto or Nigri respectively) further comprises a regulatory nucleic acidsequence, preferably a promoter region. Hence, expression of the nucleicacid encoding for the protein with recombinase activity is produced byactivating the regulatory nucleic acid sequence. Accordingly, to inducea DNA recombination, the regulatory nucleic acid sequence (preferablythe promoter region) is activated to express the gene encoding for theVika protein (or the Panto or Nigri protein respectively). Preferably,the regulatory nucleic acid sequence (preferably the promoter region) iseither introduced into the cell in the method of the invention,preferably together with the sequence encoding for Vika (or Panto orNigri respectively), or the regulatory nucleic acid sequence is alreadypresent in the cell in the beginning of the method according to theinvention. In the second case, merely the nucleic acid encoding for theVika (or Panto or Nigri respectively) protein is introduced into thecell (and placed under the control of the regulatory nucleic acidsequence).

By the term “regulatory nucleic acid sequences” within the sense of theinvention gene regulatory regions of DNA are meant. In addition topromoter regions the term encompasses operator regions more distant fromthe gene as well as nucleic acid sequences that influence the expressionof a gene, such as cis-elements, enhancers or silencers. The term“promoter region” within the sense of the invention refers to anucleotide sequence on the DNA allowing a regulated expression of agene. In this case the promoter region allows regulated expression ofthe nucleic acid encoding for Vika (or Panto or Nigri respectively).

The promoter region is located at the 5′-end of the gene and thus beforethe RNA coding region. Both, bacterial and eukaryotic promoters areapplicable for the invention.

In a method according to the invention, the vox sites (or pox sites ornox sites respectively) are either included in the cell or introducedinto the cell, preferably by recombinant techniques. This preferredmethod according to the invention further includes the steps ofintroducing into a cell the following nucleic acids:

-   a) a first nucleic acid (first recognition site, first vox site or    first pox site or first nox site respectively) comprising a nucleic    acid sequence according to or reverse complementary to SEQ ID No. 2    (or SEQ ID No. 36 or 38 respectively); or a nucleic acid sequence    that is a functional mutant thereof;-   b) a second nucleic acid (second recognition site, second vox site    or second pox site or second nox site respectively) comprising a    nucleic acid sequence identical or reverse complementary to the    nucleic acid sequence of the first nucleic acid (first recognition    site).

In a preferred method according to the invention, a nucleic acidencoding for the Vika (or Panto or Nigri respectively), protein and oneor two, preferably two, vox sites (or pox or nox sites respectively),are introduced into the cell. This method includes the following steps:

-   -   introducing into a cell the following nucleic acids:    -   i) a nucleic acid encoding for Vika (or Panto or Nigri        respectively), wherein the nucleic acid is introduced into the        DNA such, that a regulatory nucleic acid sequence (preferably a        promoter region) controls the expression of the nucleic acid        encoding for Vika (or Panto or Nigri respectively),    -   ii) a nucleic acid (first recognition site, first vox site or        first pox site or first nox site respectively) comprising a        nucleic acid sequence according to or reverse complementary to        SEQ ID No. 22 (or SEQ ID No. 36 or 38 respectively); or a        nucleic acid sequence that is a functional mutant thereof;    -   iii) a nucleic acid (second recognition site, second vox site or        second pox site or second nox site respectively) comprising a        nucleic acid sequence identical or reverse complementary to the        nucleic acid sequence defined in ii) (nucleic acid sequence of        the first recognition site), and    -   activating the regulatory nucleic acid sequence (preferably the        promoter region) to induce expression of the first nucleic acid        for the synthesis of the protein with recombinase activity.

By this preferred method according to the invention, the nucleic acidsequence encoding for Vika (or Panto or Nigri respectively), isintroduced into a cell and at least two recognition sizes (vox sites orpox sites or nox sites respectively) are introduced into the genomic orepisomal DNA of the cell. The steps i) to iii) can be performed inarbitrary order.

The introduction of the nucleic acids into the cells is performed usingtechniques of genetic manipulation known by a person skilled in the art.Among suitable methods are cell transformation bacterial cells andtransfection or viral infection for mammalian cells, whereby a nucleicacid sequence encoding the protein is introduced into the cell as acomponent of a vector or part of virus-encoding DNA or RNA. The cellculturing is carried out by methods known to a person skilled in the artfor the culture of the respective cells. Therefore, cells are preferablytransferred into a conventional culture medium, and cultured attemperatures (preferably 35-38° C.) and in a gas atmosphere that isconducive to the survival of the cells.

The method according to the invention can be performed using eukaryoticand prokaryotic cells; preferred prokaryotic cells are bacterial cells.Preferred prokaryotic cells are cells of Escherichia coli. Preferredeukaryotic cells are yeast cells (preferably Saccharomyces cerevisiae),insect cells, non-insect invertebrate cells, amphibian cells, ormammalian cells (preferably somatic or pluripotent stem cells, includingembryonic stem cells and other pluripotent stem cells, like inducedpluripotent stem cells, and other native cells or established celllines, including NIH3T3, CHO, HeLa, HEK293, hiPS). In case of humanembryonic stem cells, cells are preferably obtained without destructinghuman embryos, e. g. by outgrowth of single blastomeres derived fromblastocysts as described by (Chung 2008), by parthenogenesis, e. g. froma one-pronuclear oocyte as described by (Lin 2007) or by parthenogeneticactivation of human oocytes as described by (Mai 2007). Also preferredare cells of a non-human host organism, preferably non-human germ cells,somatic or pluripotent stem cells, including embryonic stem cells, orblastocytes.

Further, the invention includes a nucleic acid comprising a nucleic acidsequence according to or reverse complementary to SEQ ID No. 2 (or SEQID No. 36 or SEQ ID No. 38), or a nucleic acid sequence that is afunctional mutant thereof. A nucleic acid according to the inventioncomprises a maximum of 40, preferably 34, base pairs. This nucleic acidaccording to the invention includes the recognition site vox of the Vikaprotein (or the pox site of Panto or the nox site of Nigri respectively)used according to the invention. Further the invention includes a vector(also referred to herein as “reporter vector”) comprising at least onenucleic acid comprising a nucleic acid sequence according to or reversecomplementary to SEQ ID No. 2 (or SEQ ID No. 36 or SEQ ID No. 38), or anucleic acid sequence that is a functional mutant thereof. In apreferred embodiment of the invention the vector comprises at least twovox-sites (or at least two pox sites or at least two nox sitesrespectively), i.e. at least two nucleic acids that independently ofeach other exhibit a nucleic acid sequence according to or reversecomplementary to SEQ ID No. 2 (or SEQ ID No. 36 or SEQ ID No. 38), or anucleic acid sequence that is a functional mutant thereof. Thereby theat least two vox-sites are preferably not located consecutively in thevector. Rather the at least two vox sites (or at least two pox sites orat least two nox sites respectively), are positioned such that they areflanking a DNA segment, that upon recognition of the vox sites by theVika protein (or the pox sites by Panto or the nox sites by Nigrirespectively), the DNA segment is either excised or inverted. Therebythe DNA segment can preferably contain a gene or a promoter region. Asdescribed above, the DNA segment is excised when it is flanked by twovox-sites of the same orientation (same nucleic acid sequence). Aninversion of the DNA segment is catalyzed by the Vika protein (or Pantoor Nigri respectively), when the DNA segment is flanked by two vox-sites(or pox sites or nox sites respectively), arranged in oppositeorientations (i.e. comprise a nucleic acid sequence reversecomplementary to one another).

The term “nucleic acids” as used herein includes not onlydeoxyribonucleic acids (DNA) and ribonucleic acids (RNA), but also allother linear polymers in which the bases adenine (A), cytosine (C),guanine (G) and thymine (T) or uracil (U) are arranged in acorresponding sequence (nucleic acid sequence). The invention alsocomprises the corresponding RNA sequences (in which thymine is replacedby uracil), complementary sequences and sequences with modified nucleicacid backbone or 3 or 5′-terminus. Nucleic acids in the form of DNA arehowever preferred.

The term reporter vector as used herein includes a plasmid, virus orother nucleic acid carriers, that comprise a nucleic acid sequenceaccording to the invention by genetic recombination (recombinantly),e.g. by insertion or incorporation of said nucleic acid sequence.Prokaryotic vectors as well as eukaryotic vectors, for exampleartificial chromosomes, such as YAC (yeast artificial chromosomes), areapplicable for the invention. Typically, the expression vector comprisesan origin of replication, a promoter, as well as specific gene sequencesthat allow phenotypic selection of host cells comprising the reportervector.

The invention also includes a nucleic acid that encodes for a proteinwith recombinase activity, preferably Vika (or Panto or Nigrirespectively), wherein the protein preferably comprises an amino acidsequence exhibiting at least 70%, preferably at least 80%, preferably atleast 90%, particularly preferred at least 95%, even more preferred atleast 99% amino acid sequence identity to SEQ ID No. 1 (or SEQ ID No. 35or 37 respectively). Particularly preferred is a nucleic acid encodingfor the Vika protein (or Panto or Nigri respectively), used according tothe invention with a nucleic acid sequence according to SEQ ID No. 1 (orSEQ ID No. 35 or 37 respectively). Preferably, the nucleic acidcomprises a nucleic acid sequence exhibiting at least 70%, preferably atleast 80%, preferably at least 90%, particularly preferred at least 95%,even more preferred at least 99% nucleic acid sequence identity to SEQID No. 3. Furthermore, the invention includes a vector comprising saidnucleic acid according to the invention (encoding for the protein withrecombinase activity).

The invention also includes the use of the nucleic acids or vectorsaccording to the invention in a method according to the invention forproducing a site-specific DNA recombination.

When a nucleic acid encoding for a Vika protein, in particular in theform of a vector according to the invention, and at least two vox-sitesas recognition sites are introduced into a host cell, a site-specificrecombination is catalyzed upon expression of the protein Vika by itsrecognition of the vox sites.

Similar when a nucleic acid encoding for a Panto or Nigri protein, inparticular in the form of a vector according to the invention, and atleast two pox-sites or nox-sites as recognition sites are introducedinto a host cell, a site-specific recombination is catalyzed uponexpression of the protein Panto or Nigri by its recognition of the poxsites or nox sites respectively.

Accordingly, the invention also includes an isolated host cellcomprising the following recombinant DNA fragments:

-   -   at least one, preferably at least two, nucleic acids according        to the invention comprising a vox-site (preferably two nucleic        acids according to the invention that include a vox-site,        respectively, flank a further DNA segment) and/or a nucleic acid        according to the invention encoding for a Vika protein or    -   a vector according to the invention comprising at least two        nucleic acids comprising a vox-site (preferably two nucleic        acids according to the invention that include a vox-site,        respectively, flank a further DNA segment) and/or a vector        according to the invention comprising a nucleic acid encoding        for a Vika-protein.

Alternatively, the invention includes an isolated host cell comprisingthe following recombinant DNA fragments:

-   -   at least one, preferably at least two, nucleic acids according        to the invention comprising a pox-site (preferably two nucleic        acids according to the invention that include a pox-site,        respectively, flank a further DNA segment) and/or a nucleic acid        according to the invention encoding for a Panto protein or    -   a vector according to the invention comprising at least two        nucleic acids comprising a pox-site (preferably two nucleic        acids according to the invention that include a pox-site,        respectively, flank a further DNA segment) and/or a vector        according to the invention comprising a nucleic acid encoding        for a Panto-protein, or    -   at least one, preferably at least two, nucleic acids according        to the invention comprising a nox-site (preferably two nucleic        acids according to the invention that include a nox-site,        respectively, flank a further DNA segment) and/or a nucleic acid        according to the invention encoding for a Nigri protein or    -   a vector according to the invention comprising at least two        nucleic acids comprising a nox-site (preferably two nucleic        acids according to the invention that include a nox-site,        respectively, flank a further DNA segment) and/or a vector        according to the invention comprising a nucleic acid encoding        for a Nigri-protein.

The invention concerns only those isolated host cells that comprise theabove mentioned nucleic acids or vectors recombinantly and notnaturally, i.e. by genetic modification of the host cell. In particularthe invention does not include cells of the organism Vibriocoralliilyticus ATCC BAA-450 that naturally contain a nucleic acidsequence encoding for Vika and comprising the recognition site vox.Further, the invention does preferably not include cells of the organismPantoea sp. aB or Vibrio nigripulchritudo that contain a nucleic acidsequence encoding for Panto or Nigri and comprising the recognition sitepox or nox.

Particularly preferred are isolated host cells that contain both, anucleic acid encoding for Vika and at least two vox-sites (which areeither oriented in the same or in opposite direction). Further preferredare isolated host cells that contain both, a nucleic acid encoding forPanto and at least two pox-sites (which are either oriented in the sameor in opposite direction) or encoding for Nigri and at least twonox-sites (which are either oriented in the same or in oppositedirection).

A host cell within the sense of the invention is a naturally occurringcell or a (optionally transformed or genetically modified) cell linethat comprises at least one vector according to the invention or anucleic acid according to the invention recombinantly, as describedabove. Thereby, the invention includes transient transfectants (e.g. bymRNA injection) or host cells that include at least one expressionvector according to the invention as a plasmid or artificial chromosome,as well as host cells in which an expression vector according to theinvention is stably integrated into the genome of said host cell. Thehost cell is preferably selected from cells of prokaryotes andeukaryotes. Preferred prokaryotic cells are cells of Escherichia coli.Preferred eukaryotic cells are selected from yeast cells (preferablySaccharomyces cerevisiae), insect cells, non-insect invertebrate cells,amphibian cells, and mammalian cells (preferably embryonal stem cells,NIH3T3, CHO, HeLa, HEK293, hiPS). Embryonal stem cells derived bykilling of human embryos are specifically excluded from the term “hostcells” within the sense of the invention.

With the invention it is also possible to induce tissue-specificsite-specific recombination in non-human host organisms, such asmammals. Therefore the invention also includes a non-human host organismcomprising the following recombinant DNA fragments:

-   -   at least one, preferably at least two, nucleic acids according        to the invention comprising a vox-site (preferably two nucleic        acids according to the invention that include a vox-site,        respectively, flank a further DNA segment) and/or a nucleic acid        according to the invention encoding for a Vika protein or a        vector according to the invention comprising at least two        nucleic acids comprising a vox-site (preferably two nucleic        acids according to the invention that include a vox-site,        respectively, flank a further DNA segment) and/or a vector        according to the invention comprising a nucleic acid encoding        for a Vika-protein; or    -   at least one, preferably at least two, nucleic acids according        to the invention comprising a pox or nox-site (preferably two        nucleic acids according to the invention that include a pox-site        or nox-site, respectively, flank a further DNA segment) and/or a        nucleic acid according to the invention encoding for a Panto or        Nigri-protein or    -   a vector according to the invention comprising at least two        nucleic acids comprising a pox-site or nox-site (preferably two        nucleic acids according to the invention that include a a        pox-site or nox-site, respectively, flank a further DNA segment)        and/or a vector according to the invention comprising a nucleic        acid encoding for a Panto or Nigri-protein.

Explicitly included are non-human host organisms that only comprise arecombinant nucleic acid encoding for a Vika protein or Panto or Nigriprotein respectively (and that do not comprise a nucleic acid includinga vox site or pox site or nox site respectively). Furthermore, theinvention includes non-human host organisms that only comprise at leastone, preferably at least two, vox-sites or pox-sites or nox-sitesrespectively (and that do not comprise a nucleic acid encoding for aVika protein or Panto or Nigri protein respectively). In that case, atleast two vox sites (or pox site or nox site respectively), preferablyflank another DNA segment. Upon cross-breeding of two non-human hostorganisms, wherein a first host organism comprises a recombinant nucleicacid encoding for a Vika protein (or Panto or Nigri proteinrespectively) and a second host organism comprises at least tworecombinant vox-sites (or pox site or nox site respectively) andpreferably flanking a further DNA segment, the offspring includes hostorganisms expressing Vika (or Panto or Nigri respectively) and furtherincluding the recognition sites vox (or pox or nox respectively), sothat a site-specific DNA-recombination, like a tissue-specificconditional knock-out, is possible.

Non-human host organisms comprise a vector according to the invention ora nucleic acid according to the invention as described above that is,respectively, stably integrated into the genome of the host organism orindividual cells of the host organism. Preferred host organisms areplants, invertebrates and vertebrates, particularly Bovidae, Drosophilamelanogaster, Caenorhabditis elegans, Xenopus laevis, medaka, zebrafish,or Mus musculus, or embryos of these organisms.

The invention also includes a method for providing a non-human hostorganism, comprising the following steps:

-   -   providing a first non-human host organism comprising a nucleic        acid encoding for a Vika protein (preferably comprising a        nucleic acid sequence exhibiting at least 70%, preferably at        least 80%, preferably at least 90%, particularly preferred at        least least 95%, even more preferred at least 99% nucleic acid        sequence identity to SEQ ID No. 3),    -   providing a second non-human host organism comprising at least        two nucleic acids, the nucleic acid sequence of which is        independently from each other comprising a vox site (a nucleic        acid sequence according to SEQ ID No. 2) or a nucleic acid        sequence reverse complementary thereto or a nucleic acid        sequence that is a functional mutant of the aforementioned        nucleic acid sequences, wherein the at least two nucleic acids        preferably flank another DNA segment (in particular a gene or a        promoter region),    -   cross-breeding of the first and the second non-human host        organism and from the offspring obtained thereby selecting the        non-human host organisms that comprise a nucleic acid encoding        for a Vika protein and at least two nucleic acids, the nucleic        acid sequence of which is independently from each other        comprising a nucleic acid sequence according to SEQ ID No. 2 or        a nucleic acid sequence reverse complementary thereto or a        nucleic acid sequence that is a functional mutant of the        aforementioned nucleic acid sequences.

Alternatively, the invention includes a method for providing a non-humanhost organism, comprising the following steps:

-   -   providing a first non-human host organism comprising a nucleic        acid encoding for a Panto protein or a Nigri protein,    -   providing a second non-human host organism comprising at least        two nucleic acids, the nucleic acid sequence of which is        independently from each other comprising a pox site (a nucleic        acid sequence according to SEQ ID No. 36) or a nox site (a        nucleic acid sequence according to SEQ ID No. 38) or a nucleic        acid sequence reverse complementary thereto or a nucleic acid        sequence that is a functional mutant of the aforementioned        nucleic acid sequences, wherein the at least two nucleic acids        preferably flank another DNA segment (in particular a gene or a        promoter region),    -   cross-breeding of the first and the second non-human host        organism and from the offspring obtained thereby selecting the        non-human host organisms that comprise a nucleic acid encoding        for a Panto or Nigri protein and at least two pox sites (in case        of Panto, the nucleic acid sequence of which is independently        from each other comprising a nucleic acid sequence according to        SEQ ID No. 36 or a nucleic acid sequence reverse complementary        thereto or a nucleic acid sequence that is a functional mutant        of the aforementioned nucleic acid sequences) or two nox sites        (in case of Nigri, the nucleic acid sequence of which is        independently from each other comprising a nucleic acid sequence        according to SEQ ID No. 38 or a nucleic acid sequence reverse        complementary thereto or a nucleic acid sequence that is a        functional mutant of the aforementioned nucleic acid sequences).

The invention provides a novel recombinase system suitable for producinga site-specific recombination in cells of various cell types. Thereby, adiverse range of genetic manipulations can be realized, particularlyrearrangements of the DNA fragments flanked by vox sites (or pox sitesor nox sites respectively) in same orientation (excision), oppositeorientation (inversion) or when one vox site (or pox site or nox siterespectively) is present on each of two DNA molecules with one if itbeing in circular form in any orientation (integration). Exemplarymanipulations are the excision of a DNA segment that is flanked by twovox-sites (or pox sites or nox sites respectively) oriented in the samedirection mediated by the Vika (or Panto or Nigri) recombinase. Amongstothers, the recombinase systems according to the invention, inparticular the Vika/vox system, provides the possibility to excise avox-flanked (or pox-flanked or nox-flanked) stopper DNA fragment whichis located 5′ of the gene and 3′ of the corresponding to the genepromoter. Without recombination the stopper sequence prevents geneexpression, whereas upon recombinase-mediated (preferably Vika-mediated)excision of the stopper via two flanking recognition sites (preferablyvox sites) the gene is located to the proximity of the promoter andtherefore is expressed. Different types of promoter regions thatregulate the expression of the recombinase (preferably Vika) allow,inter alia, conditional DNA recombination, when for example a tissue ororganism-specific or inducible promoter region is used to express therecombinase (preferably Vika).

For the recombinase systems according to the invention, in particularthe Vika/vox system, no cross-reactivity with other recombinase systemswas observed. Therefore, the recombinase systems according to theinvention, in particular the Vika/vox system, are applicable for use incombination with other recombinase systems and becomes a particularvaluable tool for genetic experiments where multiple recombinases arerequired simultaneously or sequentially. In bacterial cells, inparticular in E. coli, a similar level of activity compared to theCre/loxP system could be demonstrated. In E. coli, the recombinasesystems according to the invention, in particular the Vika/vox system,showed a significantly higher activity than the VCre/VloxP system. Inaddition, the recombinase systems according to the invention, inparticular the Vika/vox system, is highly effective and specific inmammalian cells as well. In human and mouse cells Vika carries outsite-specific recombination on vox-sites. In mammalian cells Vika hasshown comparable activity to Cre and superior compared to VCre.Furthermore, the inventors demonstrate that Vika does not recombinepseudo-vox-sites from human or mouse genomes. As a consequence, and incontrast to Cre, there is no observed reduction in proliferation orobserved cytotoxicity upon overexpression of Vika in these cells.Therefore, the recombinase systems according to the invention, inparticular the Vika/vox system, is advantageous especially for thoseapplications in higher organisms when by using conventional SSRs such asthe Cre/lox system cytotoxicity is observed.

Due to its efficiency in a variety of cell types the recombinase systemsaccording to the invention, in particular Vika/vox, can be used inparticular for producing site-specific DNA recombination in cells inwhich other recombinase systems were shown to only achieve poor results.For example, one of the most widely used recombinase system Cre/loxP haslimited application in some model organisms such as Caenorhabditiselegans, supposedly due to the presence of loxP-like nucleic acidsequences being naturally present in the genome of C. elegans. Theinventors have shown that there is a significantly lower number ofvox-like sequences for potential targeting by Vika in the genome of C.elegans. Therefore, recombination activity of Vika/vox in C. elegans islikely to be superior to Cre/loxP.

It was demonstrated by the inventors that only low numbers of so-calledpseudo-regognition sites (pseudo-vox sites) of Vika are present in thehuman and mouse genome. The number of pseudo-vox sites is markedly lowerthan the number of pseudo-loxP sites (pseudo-recognition sites of Cre)in the mouse and human genome. It was shown, that Vika does notrecombine on pseudo-vox sites originating from human and mouse genomes.On the contrary, Cre showed prominent activity on human and mousepseudo-loxP sites.

Additionally, the inventors demonstrated in in vitro experiments, thatstable expression of Vika does not cause cytophathic effects in humanand mouse cells and that stable expression of Vika does not lead toincreased DNA damage.

The object of the invention is also solved by the use of a protein withrecombinase activity, wherein the protein comprises an amino acidsequence exhibiting at least 70%, preferably at least 80%, preferably atleast 90%, particularly preferred at least 95%, even more preferred atleast 99% amino acid sequence identity to one of the amino acidsequences according to SEQ ID No. 19, 21, 23, 25, 27, 29, 31 or 33catalyze a site-specific DNA recombination. The aforementionedsite-specific recombinase is used for site-specific DNA recombinationat, preferably two, recognition sites that are identical or reversecomplementary to each other, wherein at least one recognition sitecomprises a nucleic acid sequence according to or reverse complementaryto the nucleic acid sequence according to SEQ ID No. 20, 22, 24, 26, 28,30, 32 or 34, respectively; or a nucleic acid sequence that is afunctional mutant thereof.

The object of the invention is also solved by the use of a protein withrecombinase activity, wherein the protein comprises an amino acidsequence exhibiting at least 70%, preferably at least 80%, preferably atleast 90%, particularly preferred at least 95%, even more preferred atleast 99% amino acid sequence identity to one of the amino acidsequences according to SEQ ID No. 35 to catalyze a site-specific DNArecombination at, preferably two, recognition sites that are identicalor reverse complementary to each other, wherein at least one recognitionsite comprises a nucleic acid sequence according to or reversecomplementary to the nucleic acid sequence according to SEQ ID No. 36,respectively; or a nucleic acid sequence that is a functional mutantthereof.

The object of the invention is also solved by the use of a protein withrecombinase activity, wherein the protein comprises an amino acidsequence exhibiting at least 70%, preferably at least 80%, preferably atleast 90%, particularly preferred at least 95%, even more preferred atleast 99% amino acid sequence identity to one of the amino acidsequences according to SEQ ID No. 37 to catalyze a site-specific DNArecombination. The aforementioned site-specific recombinase is used forsite-specific DNA recombination at, preferably two, recognition sitesthat are identical or reverse complementary to each other, wherein atleast one recognition site comprises a nucleic acid sequence accordingto or reverse complementary to the nucleic acid sequence according toSEQ ID No. 38, respectively; or a nucleic acid sequence that is afunctional mutant thereof.

The proteins with recombinase activity and recognition sites are used inthe following combination (table 1). Derivatives of the respectiveprotein with amino acid sequence identities of at least 70%, preferablyat least 80%, preferably at least 90%, particularly preferred at least95%, even more preferred at least 99% and recognition sites with anucleic acid sequence that is reverse complementary to the indicatesnucleic acid sequence as well as their functional mutants are alsoincluded. It is preferred to use the indicated proteins with recombinaseactivity on wild type recognition sites or recognition sites with anucleic acid sequence reverse complementary thereto.

TABLE 1 Amino acid Nucleic acid sequence of sequence of protein (sitewild type Gen bank accession number of specific recognition protein;Organism recombinase) site EGU56467.1 SEQ ID No. 19 SEQ ID No. 20 Vibriotubiashii ATCC 19109 YP_003065675.1 SEQ ID No. 21 SEQ ID No. 22Methylobacterium extorquens DM4 YP_003280920.1 SEQ ID No. 23 SEQ ID No.24 Streptomyces sp. W9 ZP_06822377.1 SEQ ID No. 25 SEQ ID No. 26Streptomyces sp. SPB74 NP_395953.2 SEQ ID No. 27 SEQ ID No. 28Agrobacterium tumefaciens str. C58 plasmid At YP_666181.1 SEQ ID No. 29SEQ ID No. 30 Chelativorans sp_BNC1 plasmid 3 YP_957160.1 SEQ ID No. 31SEQ ID No. 32 Marinobacter aquaeolei VT8 plasmid pMAQU02 NP_943161.1 SEQID No. 33 SEQ ID No. 34 Pseudomonas sp. ND6 plasmid pND6-1WP_008927019.1 SEQ ID No. 35 SEQ ID No. 36 Pantoea sp. aB YP_004250912.1SEQ ID No. 37 SEQ ID No. 38 Vibrio nigripulchritudo

The invention is further based on the finding of the inventors that eachof the proteins listed in table 1 in column 2, shows a Crerecombinase-like activity and recognizes recognition sites as indicatedin table 1 in column 3. The organisms from which each of thesite-specific recombinases is derived from are also indicated in table 1(column 1). All of the proteins with recombinase activity listed intable 1 show a low amino acid sequence identity to Cre recombinase.

The invention further relates to a method for producing a site-specificDNA-recombination by contacting a protein with recombinase activity withthe indicated recognition sites.

The invention also includes nucleic acids exhibiting a length not morethan 40 base pairs, each nucleic acid comprising a nucleic acid sequenceaccording to SEQ ID No. 20, 22, 24, 26, 28, 30, 32 or 34 or SEQ ID No.36 or SEQ ID No.38, a nucleic acid sequence that is a functional mutantthereof or a nucleic acid sequence reverse complementary thereto.

Additionally, the invention relates to vectors comprising said nucleicacid sequences, preferably at least one, even more preferred at leasttwo identical or reverse complementary nucleic acid sequences.

In an even further embodiment the invention relates to vectorscomprising a nucleic acid encoding for a protein with recombinaseactivity wherein the protein comprises an amino acid sequence exhibitingat least 70%, preferably at least 80%, preferably at least 90%,particularly preferred at least 95%, even more preferred at least 99%amino acid sequence identity to SEQ ID No. 19, 21, 23, 25, 27, 29, 31 or33 or SEQ ID No. 35.

The use of any of the vectors according to the invention in a method forproducing a site-specific DNA-recombination is also included in theinvention.

Further, the invention includes an isolated host cell or an isolatedhost organism comprising

-   -   at least one, preferably at least two, nucleic acids according        to the invention comprising a recognition site as defined above        (preferably two nucleic acids according to the invention that        include a recognition site, respectively, flank a further DNA        segment) and/or a nucleic acid according to the invention        encoding for a protein with recombinase activity, as defined        above, or    -   a vector according to the invention comprising at least two        nucleic acids comprising a recognition site as defined above        (preferably two nucleic acids according to the invention that        include a recognition site, respectively, flank a further DNA        segment) and/or a vector according to the invention comprising a        nucleic acid encoding for a protein with recombinase activity as        defined above.

The embodiments of the invention that are described above in detail forsite-specific recombination with Vika on vox-sites and Panto on poxsites and Nigri on nox sites are also included within the invention forsite-specific recombination with one of the above mentionedsite-specific recombinases on their recognition sites.

For each of the proteins with recombinase activity, the inventorsidentified numerous putative recognition sites with a lox-like structurein the organism wherefrom the recombinase was derived. Upon extensivestudies only one thereof, the indicated respective wild type recognitionsite, turned out to be the actual recognition site of the indicatedprotein with recombinase activity. By expression of the nucleic acidsequence encoding for the site-specific recombinase in E. coli, therecombinase protein could be successfully obtained and its recombinaseactivity and specifhy for the respective wild type recognition sitecould be demonstrated. It was shown that the indicated proteins withrecombinase activity do not cross react with other known recombinasesystems and are superior in the activity compared to some of the knownrecombinase systems, at least in certain cell types.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated by the following figures andexamples without being limited to these.

FIG. 1 Amino acid sequence alignment of the sequences of Vika (SEQ IDNo. 1) and Cre (SEQ ID No. 4). Residues from Cre-recombinase known to beessential for DNA interaction are highlighted, as well as theiranalogues in the recombinase Vika, with catalytic residues in black, DNAcontacting residues underlined.

FIG. 2A Three-dimensional structure of the recombinase Vika obtained by3D-modelling.

FIG. 2B A drawing showing the crystal structure of Cre recombinase.Residues from Cre-recombinase known to be essential for DNA interactionare highlighted, as well as their analogues in the recombinase Vika,with catalytic and contacting residues.

FIG. 3 Nucleic acid sequence alignment of the recognition sites loxP(SEQ ID No. 5), rox (SEQ ID No. 9), vox (SEQ ID No. 2) and vloxP (SEQ IDNo. 11).

FIG. 4A A drawing providing a determination of specificity of theVika/vox system and analysis of potential cross-reactivities betweenVika/vox and other recombinase systems when performing DNA recombinationin E. coli. (−) and (+) indicate the addition of 100 μg/ml of L(+)-arabinose for the induction of DNA recombination. The bands for thenon-recombinant plasmids are represented as two triangles, therecombinant plasmids are represented by one triangle, M . . . DNAmarker. Specificity of the indicated site-specific DNA recombinases Cre,Dre, VCre and Vika on vox sites.

FIG. 4B A drawing providing specificity of the indicated site-specificDNA recombinases at recognition sites loxP, rox and VloxP.

FIG. 5 Determination of recombination activity of the indicatedrecombination systems Cre/loxP, VCre/VloxP and Vika/vox in E. coli. Thebands for the non-recombined plasmids are represented as two triangles,the recombinant plasmids are represented by one triangle, M . . . DNAmarker. Successful recombination events upon addition of various amountsof L (+)-arabinose for induction of DNA recombination are indicated: “noind” . . . negative control without addition of L (+)-arabinose, furtherdata show recombination upon addition of 1, 10, 100 μg/ml L(+)-arabinose as indicated.

FIG. 6A A schematic representation for determination of therecombination activity of the indicated recombination systems Cre/loxP,VCre/VloxP and Vika/vox in E. coli using a lacZ reporter assay. The lacZreporter assay; non-recombined plasmids express beta-galactosidaseresulting in the formation of blue colonies when cultured onX-Gal-containing medium; in the recombined plasmids the promoter of thelacZ gene that was originally located between two recognition sequenceswas excised, thereby beta-galactosidase is not expressed, resulting inthe formation of white colonies when cultured on X-Gal containingmedium.

FIG. 6B Photographs showing specificity of Cre, VCre and Vika inLacZ-based assay. White colonies, signifying recombination, only appearwhen a recombinase is expressed together with its correspondingreporter. Positive controls for the non-recombined (pSVpaX), andrecombined form of the reporter (pSVpaXΔ) is shown as Mock.

FIG. 7A A schematic representation with comparison of recombinationactivities of the indicated recombinase systems Cre/loxP, VCre/VloxP andVika/vox in human HeLa cells. Schematic representation of the EGFP-basedreporter assays. The non-recombined plasmids express the gene forneomycin resistance (NeoR). Upon DNA recombination, the neomycincassette is removed so that the cytomegalovirus promoter (CMV prom),that was originally driving expression of the neomycin resistance gene,is located to the proximity of the EGFP gene induces its expression.

FIG. 7B is a graph of % GFP-positive cells resulting from therecombination assays for Cre, VCre and Vika in HeLa cells.

FIG. 7C is a fluorescence microscopic image of the recombination assaysfor Cre, VCre and Vika in HeLa cells.

FIG. 8 Evaluation of genotoxicity of Vika. Recombination specificity ofVika and Cre on the respective cryptic human and mouse chromosomaltarget sites (see example 6). − and + indicates presence or absence ofL(+)-arabinose (100 μg/ml) in the growth medium. Non-recombined andrecombined plasmids are denoted as two triangles and one triangle,respectively. M, marker, 2-log DNA ladder, NEB.

FIG. 9 Evaluation of prolonged expression of Vika in mouse ES cells. A)mouse ES cell line with stably integrated Vika recombinase afterprolonged passaging (24 days). A representative photo of a clonalculture is depicted (brightfield image). B) Recombination activity ofstably expressed Vika recombinase in mouse ES cell line. Images showcells 24 hours after transfection with vox-GFP reporter plasmid. Notethe apparent Vika-mediated recombination signified through GFPexpression. A control of the recombined reporter plasmid (GFP+) wastransfected for detecting transfection efficiency.

FIG. 10 Evaluation of DNA damage induction in mouse NIH3T3 cells byγ-H2AX assay 72 h after infection with indicated recombinase-expressingvirus. Quantification of the γ-H2AX positive cells either infected withvirus for respective recombinase expression or treated withcampthothecin for 2 h. Statistically significant increase of γ-H2AXsignals is indicated by asterisks. Error bars indicate standarddeviation of the mean value. (** indicate p=0.01). n=3.

FIG. 11 Evaluation of cytotoxicity of Vika. Proliferation effects uponexpression of indicated recombinases in mouse NIH3T3 cells. Cells wereinfected with bicistronic retroviruses expressing respective recombinaselinked to GFP. Every 72 hours cells were analyzed by flow cytometry.Error bars indicate standard deviation of the mean value, n=2.

FIG. 12 Recombinase activity of Panto and Nigri on pox and noxrecombination target sites in E. coli.

EXAMPLE 1: 3D MODEL OF A PROTEIN WITH RECOMBINASE ACTIVITY (VIKA)

Vika is annotated in NCBI under number ZP_05884863 and originates fromthe gram-negative bacterium Vibrio coralliilyticus ATCC BAA-450. Vikaexhibits a low amino acid sequence identity of 27.7% to Cre recombinase(50.2% sequence similarity) (FIG. 1). In order to analyze the bindingproperties of the protein ZP_05884863 compared to Cre recombinase, a 3Datom model was developed using the crystal structure of Cre recombinaseas a template (FIG. 2). The resulting 3D model showed an RMSD(root-mean-square deviation) of 2.4±0.3 Å when compared to the Cretemplate. From this data a strong structural similarity between thesetwo proteins can be concluded. It is apparent from the 3D model thatfive catalytically important residues known from Cre recombinase areconserved in Vika.

In extensive experiments six putative recognition sites from the genomeof Vibrio coralliilyticus ATCC BAA-450 were identified by the inventors.Therefrom, the nucleic acid herein referred to as vox-site wasidentified to be the recognition site of Vika. The vox site is a 34 bpDNA sequence consisting of two inverted repeats, comprising about 50%sequence homology to loxP and 55% to VloxP and about 33% sequencehomology to rox (FIG. 3). The nucleic acid sequence of the vox-site ispresented in SEQ ID No. 2.

EXAMPLE 2: RECOMBINASE ACTIVITY OF VIKA AND RECOGNITION OF VOX-SITES INE. COLI

To verify whether the protein Vika exhibits recombinase activity and toverify that vox is its recognition site, a nucleic acid encoding forVika was cloned into an E. coli recombination reporter plasmid thatcomprised two vox-sites of the same orientation. The recombinationreporter plasmid was based on the plasmid pEVO (Buchholz and Stewart,2001), in which the recombinase was inserted via a BsrGI and Xbalcleavage site. In the plasmid, the two vox-sites flanked anapproximately 1 kb DNA segment that was excised by DNA-recombination.DNA recombination was induced by the addition of L (+)-arabinose. It wasshown that Vika mediated a DNA recombination at vox-sites (FIG. 4A,right). Therefore, it could be shown that the Vika/vox system is arecombinase system applicable in E. coli cells.

In further experiments, the activity of Vika/vox in different cell typesas well as possible cross-reactions with other recombinase systems wereassessed.

EXAMPLE 3: NO CROSS-REACTIVITY WITH OTHER RECOMBINASE SYSTEMS—NORECOGNITION OF OTHER LOX SITES BY VIKA AND NO RECOGNITION OF VOX SITESBY OTHER RECOMBINASES

To assess whether Vika recognizes the recognition sites of knownrecombinase systems, in particular loxP, VloxP and rox, the nucleic acidsequence encoding for Vika was cloned into E. coli recombinationreporter plasmids comprising the aforementioned recognition sequences,respectively. The recombination reporter plasmids were based on theplasmid pEVO (Buchholz and Stewart, 2001), into which the recombinasewas inserted via a BsrGI and Xbal cleavage sites.

In a reporter plasmid, an approximately 1 kb long DNA portion wasflanked by two lox sites of the same orientation (either loxP, VloxP orrox). Thereby upon site-specific DNA recombination (induced by additionof L (+)-arabinose) the 1 kb DNA segment was excised from the plasmid.FIG. 4B shows that the recombinase Vika is not applicable to produce aDNA recombination at any of the lox sites loxP, VloxP and rox.

Furthermore it was assessed whether other known recombinases can producea site-specific recombination on vox sites. For this purpose, reporterplasmids, each containing Cre, Dre, VCre or Vika and further includingtwo vox sites were transformed into E. coli. The results show that onlyVika, but not Cre, Dre and VCre induce recombination at vox-sites (FIG.4A). Further experiments using lacZ reporter assays confirmed thespecificity of Vika to its recognition sites vox.

Therefore, Vika can be used in combination with other recombinasesystems without causing cross-reactions.

EXAMPLE 4: ACTIVITY OF DIFFERENT RECOMBINASE SYSTEMS IN E. COLI

The activity of the recombinase systems Vika/vox, Cre/loxP andVCre/VloxP was compared in E. coli cells. For this purpose recombinationreporter plasmids as described in Example 2 or 3 that included thenucleic acid sequence encoding for the respective recombinase and tworespective recognition sites of the same orientation were used. For aquantitative analysis, different concentrations of L (+)-arabinose wereadded in order to induce the DNA recombination: 0, 1, 10 and 100 μg/ml(FIG. 5).

Further, in another approach, a lacZ reporter assay was used in order tocompare the activities of the three above-mentioned recombinase systems.For this purpose, reporter plasmids were constructed by introducing twoof the respective recognition sequences (vox, lox and VloxP) into apSV-paXl vector ((Buchholz and Bishop, 2001), schematic representationin FIG. 6A). Plasmid DNA was isolated and introduced into DH5α cells byelectroporation. The resulting cells were used in defined concentrationand plated in the presence of ampicillin on X-gal containing plates. Thenumber of white and blue colonies was counted (FIG. 6B).

In a further approach using two vectors comprising one recognition site(loxP, VloxP or vox), respectively, and the corresponding recombinases(Cre, VloxP, Vika) it was shown that only combinations of two identicaltarget sites and their corresponding recombinases produced co-integrantplasmids.

EXAMPLE 5: ACTIVITY OF DIFFERENT RECOMBINASE IN MAMMALIAN CELLS

To assess out whether the Vika/vox system is active in cells other thanthe natural host cells and E. coli, recombinase activity was analyzed inhuman HeLa cells and compared with activities of the known recombinasesystems Cre/loxP and VCre/VloxP.

For this purpose, HeLa cells were transfected with a reporter plasmidencoding for EGFP. The reporter plasmids were based on the plasmidpEGFP-X ((Buchholz and Bishop, 2001), schematic representation in FIG.7A). A neomycin cassette was flanked by two of the respectiverecognition sites of the same orientation. Using this plasmid, cloningof the respective recognition sites into said plasmid, the reporterplasmids pRK5-loxP-EGFP, pRK5-VloxP-EGFP and pRK5-vox-EGFP were derived.HeLa cells were plated in 6-well plates with a cell number of 2×10⁵cells and cultured in cell culture medium (4.5 mg/ml glucose DMEMcomprising 10% FBS and 100 U/ml penicillin/streptomycin). The reporterplasmids comprising the recognition sites (pRK5-loxP-EGFP,pRK5-VloxP-EGFP and pRK5-vox-EGFP) were co-transfected into HeLa cellswith the respective recombinase expression plasmid pNPK-NLS-Cre,pNPK-NLS-VCre and pNPK-NLS-Vika. Cells were cultured for 24 h.Subsequently, the cells were washed with PBS and fixed and examinedusing fluorescence microscopy (FIG. 7B, C).

It could be shown that the Vika/vox system is suitable for producing aDNA recombination in human HeLa cells and that it shows superioractivity when compared to the known VCre/VloxP system.

EXAMPLE 6: ACTIVITY ON CRYPTIC CHROMOSOMAL SITE OF MOUSE AND HUMANGENOMES

In order to investigate potential site-effects of Vika, pseudo-vox sitesin the mouse and human genome were identified. In both species, loweroverall numbers of pseudo-vox sites were uncovered than pseudo-loxPsites. Pseudo-vox sites that most closely resembled vox sites weretested experimentally. The nucleic acid sequences of the testedpseudo-vox sites correspond to the following SEQ ID No.:

-   -   voxCH18 (7 mutations): SEQ ID No. 12    -   voxCH21 (6 mutations): SEQ ID No. 13    -   voxCHX (6 mutations): SEQ ID No. 14    -   voxCMp92 (6 mutations): SEQ ID No. 15    -   voxCHp3 (6 mutations): SEQ ID No. 16

As a comparative example, recombination with Cre on two pseudo-loxPsites was examined. The nucleic acid sequences of the tested pseudo-loxPsites correspond to the following SEQ ID No.:

-   -   loxhXp22 (7 mutations): SEQ ID No. 17    -   loxM5 (5 mutations): SEQ ID No. 18

It was demonstrated that Cre showed prominent activity on pseudo-loxPsites (FIG. 8) in E. coli. In contrast, Vika did not display measurableactivity on pseudo-vox sites in these assays (FIG. 8).

EXAMPLE 7: PROLONGED CONSTITUTIVE EXPRESSION IN PRIMARY CELLS (MOUSE ESCELLS)

In order to examine the influence of prolonged expression of Vika inprimary cells, mouse ES cells stably expressing Vika were generated. Noeffect on cell growth and morphology was observed, indicating that Vikais active when selected for stable expression in mouse ES cells and iswell tolerated (FIG. 9).

EXAMPLE 8: DNA DAMAGE EVALUATION BASED ON γ-H2AX ASSAY

The potential impact of recombinase expression on DNA damage wasexamined. NIH3T3 cells were infected with GFP-bicistronic retroviralparticles encoding Cre, Vika or controls. Three days post infection,γ-H2AX signals were investigated in fixed cells. Cre expression caused amarked increase in γ-H2AX signals signifying induction of DNA damage. Incontrast, Vika had no influence on the amount of γ-H2AX counted (FIG.10), indicating that Vika expression does not lead to increased DNAdamage in these cells.

EXAMPLE 9: NO CYTOTOXIC EFFECT UPON HIGH-LEVEL EXPRESSION IN MOUSECELLS, (RETROVIRAL EXPERIMENT). (FIG. 11)

The percentage of GFP-positive cells over time in the populations ofexample 8 was examined as an indicator for cytotoxicity. Cre expressionled to a rapid decline of GFP positive cells in the population (that isconsistent with previously published information). However, no declinein GFP positive cells was observed in Vika expressing cells (FIG. 11).These experiments demonstrate that high levels of Cre are cytotoxic,whereas Vika expression is well tolerated in the same setting.

EXAMPLE 10: RECOMBINASE ACTIVITY OF PANTO AND NIGRI ON POX AND NOXRECOMBINATION TARGET SITES IN E. COLI

To verify whether the protein Panto and Nigri exhibit recombinaseactivity and to verify that pox and nox are corresponding recognitionsites, a nucleic acid encoding for Nigiri was cloned into an E. colirecombination reporter plasmid that comprised two nox-sites of the sameorientation. A plasmid containing gene coding for Panto protein and twoidentical sequences of pox site was created in a similar way. Therecombination reporter plasmid was based on the plasmid pEVO (Buchholzand Stewart, 2001), in which the recombinase was inserted via a BsrGIand Xbal cleavage site. In the plasmid, the two vox-sites flanked anapproximately 1 kb DNA segment that was excised by DNA-recombination(FIG. 12). DNA recombination was induced by the addition of L(+)-arabinose. It was shown that Vika mediated a DNA recombination atpox-sites Panto and Nigri on nox sites.

Therefore, it could be shown that the Panto/pox and Nigri/nox system arerecombinase systems applicable in E. coli cells.

CITED NON-PATENT LITERATURE

-   Buchholz, F., and Bishop, J. (2001). loxP-directed cloning: Use Cre    recombinase as a universal restriction enzyme. BioTechniques 31,    906-.-   Buchholz, F., and Stewart, A. F. (2001). Alteration of Cre    recombinase site specificity by substrate-linked protein evolution.    Nat Biotechnol 19, 1047-1052.-   Chung Y et al. Cell Stem Cell 2008 (2): 113-117, and supplemental    material-   Lin et al. Cell Res 2007; 17:999-1007-   Mai et al. Cell Res 2007 17: 1008-1019-   Suzuki, E., and Nakayama, M. (2011). VCre/VloxP and SCre/SloxP: new    site-specific recombination systems for genome engineering. Nucleic    Acids Research 39, e49-e49.

The invention claimed is:
 1. A method for producing a site-specificDNA-recombination, comprising: contacting a protein with recombinaseactivity inside a cell, wherein the protein has an amino acid sequenceof at least 99% amino acid sequence identity to SEQ ID No. 35, with atleast two recognition sites that are identical or reverse complementaryto each other, wherein at least one recognition site comprises thenucleic acid sequence of SEQ ID No. 36 or reverse complementary thereto,wherein upon binding of the protein with recombinase activity to the tworecognition sites, site-specific DNA-recombination occurs.
 2. The methodaccording to claim 1, further comprising: introducing into the cell anucleic acid encoding for the protein with recombinase activity.
 3. Themethod according to claim 1, wherein the cell comprises a nucleic acidencoding for the protein with recombinase activity, wherein the nucleicacid encoding for the protein with recombinase activity comprises aregulatory nucleic acid sequence and expression of the nucleic acidencoding for the protein with recombinase activity is produced byactivating the regulatory nucleic acid sequence.
 4. The method accordingto claim 1, wherein the cell is selected from eukaryotic or bacterialcells.
 5. A vector comprising at least two identical nucleic acidshaving the sequence of SEQ ID NO: 36 or a nucleic acid sequence reversecomplementary thereto, wherein the vector is a plasmid, virus, orartificial chromosome.
 6. A method for producing a site-specific DNArecombination comprising: contacting the vector of claim 5 with a cell,wherein the vector comprises a nucleic acid encoding a protein withrecombinase activity, wherein the protein comprises an amino acidsequence having at least 99% amino acid sequence identity to SEQ ID No.35, wherein upon binding of the protein with the vector, site-specificDNA-recombination occurs.
 7. The method according to claim 6, furthercomprising introducing into the cell a nucleic acid encoding for aprotein with recombinase activity.
 8. An isolated host cell or non-humanhost organism, comprising: (a) at least one nucleic acid having thesequence of SEQ ID NO: 36 or a nucleic acid sequence reversecomplementary to SEQ ID NO: 36, or (b) the vector according to claim 5;and/or a vector comprising a nucleic acid encoding for a protein withrecombinase activity wherein the protein comprises an amino acidsequence having at least 99% amino acid sequence identity with SEQ IDNO: 35.